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Phylogenetic Analysis of the Microbial Communities Associated with Exposed Gas Hydrate

Phylogenetic Analysis of the Microbial Communities Associated with Exposed Gas Hydrate. Heath J. Mills and Robert J. Martinez Dr. Patricia Sobecky’s Lab Georgia Institute of Technology. Prepare to see the following…. An introduction to the Gulf of Mexico Very important molecular techniques

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Phylogenetic Analysis of the Microbial Communities Associated with Exposed Gas Hydrate

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  1. Phylogenetic Analysis of the Microbial Communities Associated with Exposed Gas Hydrate Heath J. Mills and Robert J. Martinez Dr. Patricia Sobecky’s Lab Georgia Institute of Technology

  2. Prepare to see the following… • An introduction to the Gulf of Mexico • Very important molecular techniques • Hydrate Community Data • Bacteria Mat Community Data

  3. Gulf of Mexico Sample Sites in the Northern Gulf of Mexico Louisiana

  4. Microbial mats (Beggiatoa) Gulf of Mexico cold seep habitats

  5. Dense tubeworm beds Hydrate Drill Dense mussel beds Exposed gas hydrate

  6. DNA Extraction DNA Purification PCR Amplification Ligation and Transformation Selection Insert Not resistant to Kanamycin Lac E. coli E. coli Kanr Fails Blue/White screen Techniques used to Produce the Clone Libraries Sediment Sample

  7. PCR Amplification Cloned Insert Lac Digest the fragment using RE Topo pCR-2.1 Kanr Screen for Unique Clones

  8. 1010 109 108 Cells ml-1 107 106 105 104 Sample Sites Samples Specifics – Hydrate Community • Samples obtained from sites GC185 and GC234 • 2 cores from each site • Collected samples from three regions – • 1)sediment only • 2) Sediment/Hydrate Interface • 3) Sediment-free Hydrate • Preserved for Cell Counts, FISH and DNA/RNA extraction Cell Counts as determined by direct microscopy (cells ml-1)

  9. Methanosarcina acidovorans [M59137] Thermus Thermus aquaticus [L09663] GoM GC234 029E Green Non-Sulfur GoM GC185 523E GoM GC185 546E High G+C Actinomycetales sp. [X92695] Arthrobacter citreus [X80737] Propionibacterium acnes [M61903] Dechlorinating Clone [AF050572] Firmicutes GoM GC234 019E GoM GC234 602E Low G+C Streptococcus mutans [X58303] GoM GC234 604E GoM GC234 616E JT Clone JTB215 [AB015270] Desulfomonile tiedjei [M26635] GoM GC185 508E GoM GC234 610E Syntrophus gentianae [M26635] Delta Desulforsarcina variabilis [M34407] GoM GC185 524E GoM GC234 630E GoM GC185 515E GoM GC234 618E GoM GC185 503E GoM GC234 621E Rhodospirillum sp. [D12703] Alpha Thalassospira lucentensis [AF358664] Rhizobium leguminosarum [X67233] GoM GC234 022E Proteobacteria GoM GC234 007E Gamma Marinobacter lutaoensis [AF288157] Pseudomonas aeruginosa [U38445] Xanthomonas campestris [AF188831] Beta Burkholderia cepacia [M22518] Ralstonia pickettii [X67042] Sulfurospirillum arcachonense [Y11561] GoM GC234 615E GoM GC234 626E GoM GC185 538E GoM GC234 035E GoM GC234 005E GoM GC234 030E GoM GC234 036E Epsilon GoM GC185 507E GoM GC234 613E GoM GC234 633E Phylogenetic Analysis of the Bacteria Clone Library

  10. Methanosarcina acidovorans [M59137] High G+C Actinomycetales sp. TM56 [X92695] Arthrobacter citreus [X80737] Propionibacterium acnes [M61903] Acidaminobacter hydrogenoforma [AF016691] Clostridium elementeitii [AJ271453] Alkalliphilus auruminator [AB037677] Firmicutes GoM GC185 551S6 Clostridium halophilum [X77837] GoM GC234 019S6 Low G+C GoM GC185 608S6 G1 GoM GC234 612S6 Caloranaerobacter azorensis [AJ272422] GoM GC185 601S6 GoM GC185 503S6 Clostridium botulinum D [L37591] Anaerobacter polyendosporus [Y18189] Clostridum sp. [X98395] Desulfotomaculum ruminis [Y11572] Desulfotomaculum geothermicum [Y11567] Desulfotomaculum thermocisternum [U33455] Bacillus subtilis [AF526912] Streptococcus mutans [X58303] GoM GC185 543S6 GoM GC234 676S6 Gamma Pseudomonas aeruginosa [U38445] Bacterioplankton BB2AT2 [AY028206] Hyphomicrobium sp. Ddeep-1 [AB055793] GoM GC234 615S6 Syntrophus sp. [AJ133796] Desulfosarcina variabilis [M34407] G5 Desulfonema magnum [U45989] G3 Desulfobacterium autotrophicum [M344090] Desulfobacterium vacuolatum [M34408] G4 Desulfobacter curvatus [M34413] Desulfobacter latus [M34414] G2 Desulfobulbus sp. 3pr10 [M34411] Desulfobulbus elongates [M34413] GoM GC234 093S6 Proteobacteria HS Isolate GCA017 [AF154102] Delta GoM GC185 607S6 GoM GC234 680S6 GoM GC234 694S6 Eel-2 GoM GC234 609S6 Desulfomicrobium escambiense [AJ277886] Desulfonatronum lacustre [Y14594] G6 Desulfovibrio vulgaris [M34399] Desulfovibrio gabonensis [U31080] Desulfovibrio bastinii [U53462] Desulfovibrio halophilus [U48243] GoM GC234 051S6 GoM GC185 610S6 GoM GC185 515S6 GoM GC234 085S6 GoM GC234 023S6 Phylogenetic Analysis of the Sulfate Reducing Bacteria Clone Library

  11. Pseudomonas aeriginosa [U38445] Pyrobaculum arsenaticum str. PZ6 [AJ277124] Sulfolobus acidocaldarius [D14876] Acidianus brierleyi [X90477] Crenarchaeota GoM GC234 621R HV Clone pMC2A15[AB019718] MA Clone VC2.1[AF068822] GoM GC234 626R GoM GC234 001R Methanobacterium bryantii [M59124] Methanococcus maripaludis [AF005049] Halobacterium volcanii [K00421] SA Clone AEGEAN 71[AF290535] GoM GC234 007R GoM GC234 643R AD Clone 101B[AF268657] Thermoplasmales GoM AT425 ArD10[AY053471] GoM GC234 024R ER Clone TA1f2[AF134390] HV Clone pMC2A10[AB019739] Ferroplasma sp. MT17[AF513710] Thermoplasma volcanium str. GSS1 [299215] Methanocorpusculum parvum [M59147] SM Clone 2C174[AF015970] GoM GC234 015R Methanomicrobales GoM GC234 026R GoM GC234 008R GoM GC234 003R HV clone CS R002[AF419645] Methanogenium thermophilum [M59129] Methanogenium marisnigri [M59134] GoM GC234 633R HV clone GBa2r032[AF419632] HV clone GBa2r020[AF419631] HV clone ATR014[AF419652] ER Clone TA1a9[AF134387] ER Clone TA2e12[AF134391] GoM AT425 ArB7[AY053467] ANME-1 ER Clone BA2e8[AF134383] ER Clone Eel-36a2G10[AF354137] GoM GC234 603R GoM GC185 501R GoM GC234 614R GoM GC234 004R GoM GC234 609R GoM GC234 616R SB Clone SB-17a1A11[AF354126] SB Clone SB-17a1A2[AF354127] Methanothrix soehngenii [X16932] Methanosaeta thermoacetophila [M59141] GoM AT425 ArD7[AY053473] GoM AT425 ArB6[AY053466] GoM GC234 030R GoM AT425 ArC3[AY053469] GoM GC234 028R GoM GC234 619R B GoM GC234 033R ER Clone Eel-362A4[AF354128] A GoM GC185 517R ER Clone BA2H11fin[AF134393] SB Clone SB-24a1F10[AF354134] Methanosarcinales Methanolobus oregonensis [U20152] Methanosarcina acidovorans [M59137] D GoM GC234 606R HV Clone G72 C12[AF356643] ANME-2 GoM GC185 520R ER Clone Eel-36a2E1[AF354139] ER Clone TA1a4[AF134384] GoM GC185 505R GoM GC234 009R GoM GC234 622R C GoM GC234 634R GoM GC185 503R GoM AT425 ArD2[AY053472] GoM GC234 021R ER Clone Eel-36a2A1[AF354129] GoM GC234 607R GoM GC234 019R HV Clone C1 R019[AF419638] SB Clone SB-24a1A12[AF354131] ER Clone Eel-36a2A5[AF354133] Phylogenetic Analysis of the Archaea Clone Library

  12. Phylogenetic Analysis of the ANME Clone Library Pseudomonas aeruginosa [U38445] Pyrobaculum arsenaticum str. PZ6 [AJ277124] Crenarchaeota Acidianus brierleyi [X90477] Sulfolobus acidocaldarius [D14876] Methanococcus maripaludis [AF005049] Methanobacterium bryantii [M59124] GoM Clone AT425 ArD10 [AY053471] GoM GC234 011A Thermoplasmales GoM GC185 601A GoM GC234 045A Ferroplasma sp. MT17 [AF513710] Thermoplasma volcanium str. GSS1 [AJ299215] Halobacterium volcanii [K00421] Methanocorpusculum parvum [M59147] GoM GC185 624A GoM GC234 033A GoM GC234 022A GoM GC234 633A Methanomicrobales Methanogenium organophilum [M59131] Methanogenium frigidum [AF009219] Methanogenium thermophilum [M59129] GoM GC234 009A Methanospirillum hungatei [M60880] ANME-1 GoM GC234 610A Methanosaeta thermoacetophila [AF334401] Methanothrix soehngenii [X16932] Methanosaeta sp. [AJ133791] B A C Methanosarcinales ANME-2 Methanolobus oregonensis [U20152] Methanosarcina acidovorans [M59137] GoM GC185 618A D GoM GC185 623A GoM GC234 046A GoM GC185 626A GoM GC234 677A GoM GC185 620A

  13. Data Summary – Hydrate Community • Samples acquired using the Johnson Sea Link manned submersible • Over 120 clones obtained from 16S rDNA sequences specific for Bacteria, Archaea, Sulfate Reducing Bacteria, and Anaerobic Methane Oxidizing Bacteria • Numerically dominant Bacteria clones obtained from the Delta and Epsilon classes of the phylum Proteobacteria • Majority of Archaea clones were related to the previously identified ANME-1 and -2 clusters • Two new clusters of clone identified in the Anaerobic Methane Oxidizing bacteria library • Preliminary Data – • Very little over lap between the sediment, sediment/hydrate interface, and the sediment free hydrate for either Bacteria or Archaea libraries • Dominant Bacteria – Delta • Dominant Archaea – ANME-2

  14. Samples Specifics – Mat Community • Samples obtained from sites GC185 and GC234 containing either Orange or White Beggiatoa visible on sediment surface • 2 cores from each site • Collected samples from three regions – • 1) 0-2 cm • 2) 6-8 cm • 3) 10-12 cm • Preserved for Cell Counts, FISH and RNA extraction

  15. Data Summary – Mat Community • Over 340 clones from Bacteria and Archaea libraries where analyzed. • The Archaea library was dominated by anaerobic methane oxidizing clones related to the ANME-2 cluster • The Bacteria library was dominated by Deltaproteobacteria at the lower sediment levels and shared numerical significance with Gammaproteobacteria at the 0-2 cm depth. Total Archaea Library Total Bacteria Library

  16. Dr. Patricia Sobecky Lab Members Cassie Hodges Kristen Wilson Committee Members Dr. Roger Wartell Dr. Thomas DiChristina Dr. Joe Montoya Dr. Frank Loeffler Project Collaborators Dr. Ian McDonald Dr. Samantha Joye Acknowledgements Crew of the R/V Seward Johnson I and II

  17. Samples Specifics – Mat Community

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